Hemibiotrophic secretome of the anthracnose pathogen Colletotrichum truncatum. V.BHADAURIA, S. BANNIZA AND Y. WEI. Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada; and (S.B.) Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Colletotrichum truncatum (Schwein.) Andrus & Moore causes anthracnose disease of many leguminous species, including lentil (Lens culinaris Medik.), soybean (Glycine max (L.) Merrill), fababean (Vicia faba L.) and pea (Pisum sativum L.). This fungal pathogen employs a two-stage, hemibiotrophic (biotrophic and hemibiotrophic) infection strategy to colonize lentil plants. The switch from biotrophy to necrotrophy is critical in disease/symptom development. During this transition, C. truncatum secretes large repertoires of proteins, which might be instrumental in switching the fungus from biotrophic (characterized by intracellular primary hyphae) to necrotrophic phase (characterized by thin secondary hyphae). To catalogue these secretory proteins, we constructed a directional cDNA library from a susceptible lentil cultivar ‘Eston’ infected with C. truncatum isolate CT-21 at switch point. In silico analysis of 5000 expressed sequence tags (ESTs) predicted that 57% of the ESTs were likely to encode proteins of plant origin and 39% of fungal origin. The remaining 4% ESTs were considered to be of unknown origin. This directional cDNA library allowed us to scan fungal ESTs for canonical N-terminal signal peptides by 5'-end single pass sequencing. Around 10% predicted open reading frames from these fungal ESTs were likely to encode proteins with N-terminal signal peptides. We refer to these proteins as secreted proteins and collectively as hemibiotrophic secretome. Using ContigExpress software (Invitrogen), these fungal ESTs could be assembled into 31 contigs and 105 singletons/singlets, giving a total of 136 unigenes. The unigenes were then queried against the NCBI non-redundant protein database using BLASTX. Identified secreted proteins were classified into four groups: cell wall associated proteins, cysteine rich proteins, hydrolases (cell wall degrading enzymes and proteases), and others. Semi-quantitative RT-PCR was performed to verify the expression of secreted proteins in a time-course study at transcriptome level. Furthermore, we selected three secreted proteins (putative elicitors) for heterologous expression in Pichia pastoris (Guillierm.) Phaff, which delineated that these proteins induce HR cell death in tobacco leaves after infiltration into leaf mesophyllum.
Functional groups of microfungi associated to roots of willows grown under short-rotation intensive culture. A. H. CORREDOR, K. VAN REES AND V. VUJANOVIC. Department of Food and Bioproduct Sciences; and (K.V.) Department of Soil Science, University of Saskatchewan, College of Agriculture and Bioresources, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Short-rotation intensive cultures (SRIC) of willows (Salix spp.) are promising alternatives for biomass and bioenergy production in Canada. Despite the physiological plasticity of willows under natural conditions, their establishment in SRIC systems is challenging due to important disease outbreaks. The objective of this study was to identify potentially pathogenic and beneficial fungi found in association to roots of willows grown under SRIC in Alberta and Saskatchewan. Sampling of roots of healthy and diseased plants resulted in isolation and identification of 12 potentially beneficial and nine potentially pathogenic fungi. Potentially pathogenic fungi were tested on apples to determine their ability to induce necrosis. Based on these results as well as their abundance in roots of diseased plants, three potential pathogenic fungi were selected. Chosen potentially pathogenic fungi were identified as Fusarium culmorum (Smith) Sacc., Didymella bryoniae (Fuckel) Rehm, and Neonectria radicicola (Gerlach & Nilsson) Mantiri & Samuels. Beneficial isolates were tested in co-culture with each pathogenic isolate. Eight of these fungi exhibited the ability to limit pathogen growth. Ongoing experiments will test the ability of these fungi to control all pathogenic fungi in planta. Overall results of this study will identify fungal diseases that may arise when using previously arable land for willow SRIC. Additionally, this work will enlighten the prospective use of native fungi to contribute in the establishment of willow SRIC in Canadian prairies.
Sphaerodes quadrangularis as biotrophic mycoparasitic fungus on pathogenic Fusarium avenaceum. Y. K. GOH AND V. VUJANOVIC. Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (V.V.) Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Sphaerodes quadrangularis García, Stchigel & Guarro is a facultative, contact biotrophic mycoparasite of Fusarium avenaceum (Fr.) Sacc. (dominant fusarium head blight pathogen in Western Canada). Sphaerodes quadrangularis establishes an intimate relationship with Fusarium host, producing hook-shaped and clamp-like structures or branches deriving nutrients and other essential factors from living host cells. Mycoparasite's contact cells surround, either partially or completely, and then envelop the host hyphae. Contact parasitic structures, although not penetrating Fusarium hyphae, demonstrate the ability to reduce the size of parasitized host hyphal cells. Other Fusarium species, such as F. oxysporum Schlecht.:Fr., F. equiseti (Corda) Sacc., F. poae (Peck) Wollenw., F. arthrosporioides Sherb., F. flocciferum Corda, F. torulosum (Berk. & Curt.) Nirenberg, F. culmorum (Smith) Sacc., and a toxigenic F. graminearum Schwabe chemotype, did not trigger mycoparasitic response or formation of S. quadrangularis specialized attachment structures, showing specificity of S. quadrangularis for F. avenaceum.
Interaction between pathogenic and beneficial fungi in willow phyllosphare. Z. HOSSEINI-NASABNIA, K. VAN REES AND V. VUJANOVIC. Department of Food and Bioproduct Sciences; and (K.V.) Department of Soil Science, University of Saskatchewan, College of Agriculture and Bioresources, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
In this study, fungal diversity of healthy and diseased willow in Canadian (SK, AB and MB) short-rotation intensive cultures (SRIC) plantations was assessed. A total of 106 fungal taxa originating from stem and leaf as well as 79 from cuttings were isolated and characterized using molecular taxonomy and phylogeny. Fungal communities associated with healthy and diseased willow depicted conductive ecological parameters, which were used to calculate fungal dominance and persistence indices. Potential pathogenic fungi, belonging mostly to Ascomycota and Basidiomycota, were tested using apple artificial inoculation bioassays investigating fungal ability of producing necrotic lesions on plant tissues. Diaporthe eres Nitschke, Cadophora luteo-olivacea (Beyma) Harr. & McNew, Leucostoma niveum (Hoffm.) Höhn., Sclerotinia spp., Protoventuria alpina (Sacc.) Barr, Truncatella angustata (Pers.) Hughes and Glomerella cingulata (Stoneman) Spauld. & Schrenk showed important virulence levels. Among 13 promising beneficial fungi tested in dual-culture plate assay for their biocontrol/bioprotection ability, six showed efficient suppression of pathogen growth. In planta assays are currently underway to evaluate pathogenic fungi effects during willow growth phenophases under greenhouse conditions. Current results support findings that major pathogenic fungal isolates may originate from willow-cutting or previous land crops. Results are expected to have a significant impact on fungal pathogen management strategies in willow SRIC.
Genetic analyses of crown rust resistance in Avena sterilis accessions. Y. LIN, C. A. MCCARTNEY AND J. CHONG. Crop Development Centre, Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada; and (J.C.) Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada
Crown rust, caused by Puccinia coronata Corda f. sp. avenae Eriks., is the most economically important disease for cultivated oat (Avena sativa L.) in western Canada. Resistant oat varieties are an important control for crown rust. The development of resistant varieties requires the identification of new resistance genes because crown rust resistance genes are typically not durable. Avena sterilis L. is a major source of crown rust resistance genes. AC Morgan (Reg. no. CV-369, PI 629113) is a well-adapted, high-yielding and lodging-resistant cultivar, but it is highly susceptible to crown rust. The objective was to discover new effective crown rust resistance genes in A. sterilis accessions. Four genetic populations were generated by crossing AC Morgan with six A. sterilis accessions. F2 populations were inoculated with isolate CR259 (highly virulent on Pc 59 and Pc91) at the two-leaf stage and rated for disease reaction 11 and 13 days post-inoculation. Chi-square analysis of F2 data indicated single gene control of resistance in PI375506 (3R: 1S) and PI375547 (3R:1S), and two-gene control of resistance in PI333561 (15R:1S), PI311623 (15R:1S). Evaluation of F3 families will be conducted to confirm these results. These preliminary data suggest that putative new effective crown rust resistance genes have been identified for use in oat breeding.
Effect of microbial and synthetic fungicides in combination with resistant crop cultivars for managing clubroot, 2009. G. PENG, M. R. MCDONALD, S. F. HWANG, B. D. GOSSEN AND S. E. STRELKOV. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (M.R.M.) Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph ON N1G 2W1, Canada; (S.F.H.) Crop Diversification Centre North, Alberta Agriculture and Rural Development, 17507 Fort Road NW, Edmonton, AB T5Y 6H3, Canada; and (S.E.S.) Department of Agricultural, Food and Nutritional Science, University of Alberta, 410 Agriculture/Forestry Centre, Edmonton, AB T6G 2P5, Canada
Clubroot, caused by Plasmodiophora brassicae Woronin, is a major disease of crucifer vegetable crops in eastern Canada and an emerging threat to canola (Brassica napus L.) production in western Canada. Resistant cultivars of canola and other crops have recently become available, but they are not immune to clubroot and the durability of this resistance is not known. In previous studies, several microbial and synthetic fungicides reduced clubroot severity under controlled conditions. The current study was conducted to evaluate the efficacy of these products on resistant (R) and susceptible (S) cultivars, in two canola field trials near Edmonton, Alberta and one canola trial and one Chinese cabbage (Brassica rapa L. subsp. Chinensis (Rupr.) var. utilis Tsen and Lee) trial near Bradford, Ontario in 2009. The study sites were naturally infested with P. brassicae. Each plot consisted of four 6 m long rows. The microbial fungicides Serenade AOS (Bacillus subtilis (Ehrenberg) Cohn strain QST 713) and Pre-Stop (Gliocladium catenulatum Gilman & Abbott strain J1446), and synthetic fungicides Ranman (cyazofamid) and Allegro 500F (fluazinam) were sprayed in 500 L water ha−1 into furrows at the following rates: 10 L, 1.4 Kg, 0.54 L, and 2.9 L, respectively, before covering the furrows with soil. Plants (20–25) from the centre 4 m section of each plot were examined for clubroot severity at full bloom (canola) or eight weeks after seeding (Chinese cabbage). R canola cultivars reduced clubroot severity by 87–93% relative to S cultivars, and the R Chinese cabbage cultivar reduced severity by 99%. Severe early-season drought in Alberta, and poor S canola cultivar germination compounded with uneven pathogen distribution in Ontario inhibited assessment of fungicide efficacy in these canola trials. On Chinese cabbage, however, all four fungicides reduced severity on the S cultivar, lowering the disease severity by 50–85% compared with nontreated S cultivar controls.
Characterization of Stagonospora nodorum isolates at microsatellite, mating type and toxin loci. M. STEGMANN, R. STONEHOUSE AND C. A. MCCARTNEY. Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Stagonospora nodorum (Berk.) Castellani & Germano (teleomorph Phaeosphaeria nodorum (Müller) Hedjaroude) is the causal agent of stagonospora nodorum blotch in wheat (Triticum aestivum L.), which is an important disease in Saskatchewan and globally. Genetic analyses of the basis of host–pathogen interactions in this pathosystem will provide information useful in the development of resistant wheat varieties. The objectives of this project were to assess genetic variability among S. nodorum isolates, assess the contribution of sexual reproduction to population genetics, and characterize isolates for future genetic analyses. To accomplish this goal, 71 S. nodorum isolates from western Canada were characterized with expressed sequence tag (EST)-derived microsatellite loci (SNOD), mating type specific primers (MAT1-1 and MAT1-2), and host-selective toxin (HST) specific primers (SnToxA and SnTox3). The isolates showed an equal distribution of the two mating type idiomorphs, which would support sexual reproduction. Data collection on the EST-derived microsatellite loci (SNOD) is underway, and will determine whether sexual reproduction is occurring. Comparisons will be made between the presence of specific HSTs and isolate virulence.
Novel pyrenomycetous fungal and Bacillus- like strains-potential biocontrol agents against 3-AcetylDeoxynivalenol-producing Fusarium graminearum on wheat and barley. V. VUJANOVIC, Y. K. GOH AND M. B. MANSOUR. Department of Food and Bioproduct Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, Canada; and (M.B.M.) Department of Biology, University of Tunis, Tunis 1060, Tunisia
Among Fusarium spp., Fusarium graminearum Schwabe strains are the most aggressive and well-known causal agents of fusarium head blight (FHB) and other Fusarium-associated plant diseases in Saskatchewan and Canada. Fusarium graminearum 3-acetyldeoxynivalenol (ADON) chemotype is spreading to western Canada, replacing dominant Fusarium strains. Our goal is to evaluate two potential microbial biological control candidates, one pyrenomycetous fungus SMCD 2250 and Bacillus-like bacterium strain SMCD B12 for their ability to control FHB disease caused by F. graminearum 3-ADON (SMCD 2243) on wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), in greenhouse trials. Wheat and barley inoculated with pyrenomycetous fungus SMCD 2250 and Bacillus-like SMCD B12 showed an increase in height, number of spikes, number of plants per pot, and weight of spikes produced compared with control setup. Antibiosis and inhibition mechanisms are most likely employed by these microbial isolates to control or suppress F. graminearum, suggesting potential agricultural applications in managing FHB disease on wheat and barley. In the current study, seeds obtained from both hosts will be subjected to high performance liquid chromatography to analyze 3-ADON mycotoxin accumulation in the presence of biocontrol agents. Also, using F. graminearum-specific primer sets and quantitative real-time polymerase chain reaction, F. graminearum DNA presence in spikes will be measured.